Knowledge of the detailed three-dimensional structure of any given macromolecule is critical for optimizing and/or regulating the use of that macromolecule, be it a protein that is malfunctioning in a metabolic pathway, or a synthetic polymer used in microchip technology. Currently, there are two major strategies for determining the detailed three-dimensional structure of a macromoleucle: X-ray crystallography and nuclear magnetic resonance. X-ray crystallographic analysis requires the time-consuming process of preparing high quality crystals, whereas classical NMR three-dimensional analysis is limited to macromolecules that are under 35 kilodaltons [Yu, Proc. Nat. Acad. Sci. USA 96:332-334 (1999)]. Furthermore, such methods of high-resolution structure determination are generally applicable to macromolecules formed by tight contacts between the individual, well-structured components of the macromolecule. These methods have more limited applicability in those cases where there are weaker interactions between the component; examples include the relatively transient associations formed in complexes involved in signal transduction, or in transcriptional control. Crystal structures of such complexes might be biased by packing forces comparable to the interdomain interactions, while the precision and accuracy of the conventional NMR structural approaches are necessarily limited by the restricted number of nuclear Overhauser effect (NOE) contacts and by interdomain flexibility rendering the available NOE information uninterpretable.
Recently proposed NMR approaches [Tolman et al., Proc. Natl. Acad. Sci., U.S.A., 92:9279-83 (1995); Bruschweiler et al., Science, 268:886-9 (1995); Broadhurst et al., Biochemistry, 34:16608-17 (1995); Tjandra et al., Nat. Struct. Biol., 4:443-9 (1997); and Tjandra and Bax, Science, 278:1111-4] are potentially capable of improving both the accuracy and precision of structure determination in solution and might prove to be the method of choice in those cases when the number of available short-range NOE contacts is limited. These methods are based on `long-range` structural information in the form of inter-nuclear vector constraints with respect to an overall, molecular reference frame. These constraints may arise from correlation with the anisotropic hydrodynamic properties of the molecule [Bruschweiler et al., Science, 268:886-9 (1995); Broadhurst et al, Biochemistry, 34:16608-17 (1995); and Tjandra et al, Nat. Struct. Biol., 4:443-9 (1997)], or from weak alignment of molecules in solution caused by either their interaction with the magnetic field [Tolman et al., Proc. Natl. Acad. Sci, U.S.A., 92:9279-83 (1995)] or by the liquid crystalline characteristics of the medium [Tjandra and Bax, Science, 278:1111-4]. The NMR relaxation approach [Bruschweiler et al., Science, 268:886-9 (1995); Broadhurst et al., Biochemistry, 34:16608-17; and Tjandra et al, Nat. Struct. Biol., 4:443-9 (1997)] which takes advantage of the anisotropic character of the overall rotation, is most generally applicable to a wide range of macromolecules in their native milieu. The magnetic alignment method [Tolman et al., Proc. Natl. Acad. Sci., U.S.A., 92:9279-83 (1995) and Tjandra et al., Nature Structural Biology, 4:732 (1997)] requires macromolecules to possess a sufficiently high anisotropy of the magnetic susceptibility, and is not, therefore, widely applicable. The approaches based on weak alignment of macromolecules in liquid crystalline medium may be restricted by possible interactions between the molecule under investigation and the medium. For a list of intractable target proteins by this method using lipid bicelles see footnote 8 in Clore et al.,[ J. Am. Chem. Soc., 120:10571-2 (1998)], although more recent alignment methods may alleviate this issue [Clore et al., J. Am. Chem. Soc., 120:10571-2 (1998); Hansen et al., J. Am. Chem. Soc., 120:11210-11 (1998); Koenig et al., J. Am. Chem. Soc., 121:1385-6 (1999); and Sass et al., J. Am. Chem. Soc., 121:2047-55 (1999)].
Naturally occurring polymers such as nucleic acids and proteins are macromolecules that have distinct three-dimensional structures. Indeed, the ability of any given protein to carry out its physiological role, regardless of whether it functions as a structural element, a binding partner, and/or a biochemical catalyst, requires that the protein assume a specific conformation. This conformation is dependent on the three-dimensional folding of the protein into specific domains and the orientation of these domains to each other, as well to the corresponding domains of other proteins.
The binding of a ligand to a protein (e.g., a substrate to an enzyme), generally results in a local alteration of the three-dimensional structure of the protein. In addition, the binding of the ligand to one site of a protein, can also alter the structure of other regions of the polypeptide [See generally, Kempner, FEBS 326:4-10 (1993)]. Indeed the relative orientation and motions of domains within many proteins are key to the control of multivalent recognition, or the assembly of protein-based cellular machines. Therefore, it is not surprising that there has been a long and continuous effort to determine the structures of nucleic acids and proteins, not only in their resting state, but also in their more dynamic state in their native environment.
In recent years it has become apparent that there is a large but finite number of protein structural domains that are shared throughout nature. These domains are used by the proteins to carry out their biological roles. One such pair of domains are the src homology domains SH2 and SH3. Eukaryotic cellular signal-transduction pathways that are initiated by transmembrane receptors with associated tyrosine kinases rely on these two small protein domains for mediating many of the protein-protein interactions that are necessary for transmission of the signal [Cantley et al., Cell 64:281-302 (1991); Schlessinger et al., Neuron 9:383-391 (1992); Pawson et al., Curr. Biol. 3:434-442 (1993)]. These domains were first discovered in cytoplasmic (non-receptor) protein tyrosine kinases such as the src oncogene product, thus leading to the term `src homology domains` [Sadowski et al., Mol. Cell. Biol. 6:4396-4408 (1986)].
The unique importance of these domains became clear with the discovery of the crk oncogene product, which consists of little more than an SH2 and an SH3 domain fused to the viral gag protein, but is capable of transforming cells [Mayer et al,. Nature 332:272-275 (1988)]. SH2 and SH3 domains have been identified in molecules with distinct functions that act downstream from the receptors for, among others, epidermal growth actor (EGF), platelet-derived growth factor (PDGF), insulin and interferon, and the T-cell receptor [Koch et al., Science: 252:668-674 (1991)].
An important aspect of the role of protein domains such as the SH2 and SH3 domains is their ability to recognize particular amino acid sequences in their target proteins: SH2 domains bind tightly to phosphorylated tyrosine residues [Anderrson et al., Science; 250:979-982 (1990); Matsuda et al. Science 248:1537-1539 (1990); Moran et al. Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990); Mayer et al Proc. Natl. Acad. Sci. USA: 88:627-631 (1991); Songyang et al. Cell 72:767-778 (1993)] whereas SH3 domains bind to proline rich segments forming a short helical turn in the complexes [Kuriyan and Cowburn, Annu. Rev. Biophys. Biomol. Struct., 26:259-288 (1997), the contents of which are hereby incorporated herein by reference in its entirety]. The modular nature of these domains is made clear by the fact that they occur in different positions in the polypeptide chains of the intact proteins of which they are a part, and that the binding functions can often be reproduced by isolated domains. Although SH2 and SH3 domains frequently occur close together in sequence, some proteins have only one or the other domain, and some have more than one version of either domain. Proteins that contain more than one of these domains do not always maintain a strict spacing or particular order between the domains.
Even for the SH2 and SH3 domains for which the individual structural properties and ligand specificities are fairly well understood, the structural organization and interactions between them in the multidomain complexes are complex, and difficult to elucidate. These interactions are likely to be of significance, in particular, in view of the frequency of protein constructs containing adjacent SH2 and SH3 domains. Examples of structural studies of the multiple SH3/SH2 domain constructs are the Abelson protein tyrosine kinase SH(32) or Abl SH(32); Lck SH(32); Grb2 SH(323); Hck SH(321); Src and Src SH(32) [reviewed in Kuriyan and Cowburn, Annu. Rev. Biophys. Biomol. Struct., 26:259-288 (1997); and Sicheri and Kuriyan, Curr. Opin. Str. Biol., 7:777-785 (1997)]. Structural approaches to these complexes are complicated by the limited contacts and energies of the interdomain interactions. While the crystal structures of the src-family SH(321) kinase systems have shed significant insight into unexpected kinases/SH3 interactions, and demonstrated the allosteric nature of kinase inhibition by intramolecular phosphorylation, these structures of the down-regulated, inactive forms of the enzymes do not provide a detailed understanding of the mechanism of regulation, or the roles of domains in substrate recognition [Sicheri and Kuriyan, Curr. Opin. Str. Biol., 7:777-785 (1997) and Mayer et al., Current Biology, 5:296-305 (1995)]. This issue of interdomain flexibility in solution is a general one for large multidomain proteins [Campbell and Downing, Nat. Struct. Biol., 5 Suppl.:496-9 (1998)].
Therefore, there is a need for determining the structural organization and interactions of components of macromolecules including monitoring enzymatic reactions, DNA-protein interactions, ligand binding, and protein folding. Furthermore, there is a need to exploit such determinations in order to be able to design more potent drugs, pharmaceutical therapies and diagnostic agents. In addition, there is a need to further elucidate the complex structural characteristics of synthetic chemical polymers in solution.
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